ABSTRACT
This paper compared the differences between two kinds of Bufonis Venenum produced by Bufo gargarizans gargarizans and B. gararizans andrewsi, and verified the rationality of the market value orientation of Bufonis Venenum based on the zebrafish mo-del. Twenty batches of Bufonis Venenum from Jiangsu province, Hebei province, Liaoning province, Jilin province, and Liangshan, Sichuan province, including B. gargarizans gargarizans and B. gararizans andrewsi, were collected. The UHPLC-LTQ-Orbitrap-MS combined with principal component analysis was used to compare the differences between two kinds of Bufonis Venenum. According to the limiting conditions of VIP>1, FC<0.5 or FC>2.0, and peak total area ratio>1%, 9 differential markers were determined, which were cinobufagin, cinobufotalin, arenobufagin, resibufogenin, scillaredin A, resibufagin, 3-(N-suberoylargininyl)-arenobufagin, 3-(N-suberoylargininyl)-marinobufagin, and 3-(N-suberoylargininyl)-resibufogenin. The content of 20 batches of Bufonis Venenum was determined according to the Chinese Pharmacopoeia(2020 edition) by high-performance liquid chromatography, and the 2 batches of Bufonis Venenum, CS7(8.99% of total content) and CS9(5.03% of total content), with the largest difference in the total content of the three quality control indexes of the Chinese Pharmacopoeia(bufalin, cinobufagin, and resibufogenin) were selected to evaluate their anti-liver tumor activity based on the zebrafish model. The tumor inhibition rates of the 2 batches were 38.06% and 45.29%, respectively, proving that only using the quality control indexes of the Chinese Pharmacopoeia as the value orientation of Bufonis Venenum market circulation was unreasonable. This research provides data support for the effective utilization of Bufonis Venenum resources and the establishment of a rational quality evaluation system of Bufonis Venenum.
Subject(s)
Animals , Zebrafish , Bufanolides/analysis , Bufonidae , Chromatography, High Pressure Liquid , Quality Control , Cell Line, TumorABSTRACT
ObjectiveTo develop a quantitative analysis of multi-components by single marker （QAMS） for determination of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills， and to provide a method for improving the national standard of the pills. MethodHigh performance liquid chromatography （HPLC） was developed for simultaneous determination of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills and the methodology validation was carried out. The chromatographic separation was performed on a Nucleosil 100-5 C18 column （4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile -0.1% potassium dihydrogen phosphate aqueous solution （pH adjusted to 3.2 with phosphoric acid） （48∶52）， and the flow rate was 0.6 mL·min-1， the detection wavelength was set at 296 nm and the column temperature was 35 ℃. Taking cinobufagin as the internal standard， the relative correction factors （RCFs） of bufalin and resibufogenin were calculated， and the key influencing factors of RCFs were investigated. Relative retention time was used for the chromatographic peak location of the analyte， combining with the on-line ultraviolet spectroscopy and accurate relative molecular weight obtained by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-QTOF/MS）. The external standard method was used to verify the contents of three components obtained by QAMS. ResultQAMS was established for the determination of bufalin， cinobufagin and resibufogenin in the samples， and RCFs of cinobufagin to bufalin and resibufogenin were 0.922 and 1.01， respectively. The total content of the three marker compounds in 11 batches of Shexiang Baoxin pills was 33.7-36.0 µg per pill. There was no significant difference between the quantitative results of QAMS and external standard method. ConclusionThe established method can be used for the quality control of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills. It is suggested that bufalin should be considered as one of three marker compounds， and the sum of bufalin， cinobufagin and resibufogenin should be used for the content limit of this preparation.
ABSTRACT
As a new strategy capable of uncovering the characteristics of traditional Chinese medicines, the quantitative analysis of multi-components by single-marker(QAMS) has been widely employed for the quality evaluation of Chinese medicinal materials, slices, and extracts. However, its application in the assessment of Chinese patent medicines is yet to be explored. By referring to the determination of three bufogenins in Bufonis Venenum by QAMS described in Chinese Pharmacopoeia(2020 Edition), this paper selected seven representative preparations containing Bufonis Venenum and explored whether the relative correction factors(RCFs) of cinobufagin(CB) to bufalin(BF) and resibufogenin(RB) could be directly used for the quality control of Bufonis Venenum-contained preparations. Based on the qualitative analyses under the same chromatographic conditions as used for toad venom, combing specificity test, five preparations such as Yatong Yili Pills, Houzheng Pills, Xiongdan Jiuxin Pills, Liushen Pills and Niuhuang Xiaoyan Pills, were expected to use validated RCFs for the direct determination of three components. Taking Houzheng Pills as an example, the methodological validation of bufalin, cinobufagin and resibufogenin was carried out, and the recoveries of bufalin, cinobufagin and resibufogenin were 90.64%-106.1%. The obvious difference was not observed between the contents of bufalin and resibufogenin in 24 batches of preparation samples by QAMS and external reference method. In the tested samples, the content of bufalin, cinobufagin and resibufogenin were 1.27-2.61, 2.44-5.66 and 0.988-3.16 mg·g~(-1) in 10 batches of Liushen Pills samples. The contents of bufalin, cinobufagin and resibufogenin were 0.760-1.32, 1.35-2.39 and 0.600-1.55 mg·g~(-1) in 10 batches of Houzheng Pills samples from three manufacturers. The obtained data contribute to improving the quality standard of Bufonis Venenum-contained preparations, and they also provide some ideas for the application of QAMS in the quality evaluation and control of Chinese patent medicines.
Subject(s)
China , Chromatography, High Pressure Liquid , Medicine, Chinese Traditional , Nonprescription Drugs , Quality ControlABSTRACT
Bufonis Venenum,the dried secretion of Bufo bufo gargarizans or B. melanostictus,is toxic and hard with the efficacy of removing toxicity for detumescence and relieving pain. The processing of Bufonis Venenum dates back to the Song dynasty. In addition to the wine-processing,milk-processing and talcum powder-processing,there were some other kinds of processing methods in ancient times,such as baking,calcining,water-soaking and vinegar-processing. Modern studies have shown that the Bufonis Venenum has the main chemical components of bufadienolides,indole alkaloids sterols,and other compounds. It has the pharmacological effects of antitumor,cardiac,antibacterial,and analgesic activities,local anesthesia,and so on. This paper reviews the processing evolution,chemical components and pharmacological effects of Bufonis Venenum,providing references for its special processing and modern research as well as the theoretical basis for the research on its processing mechanism and quality control.
Subject(s)
Animals , Bufanolides/pharmacology , Bufonidae , Quality ControlABSTRACT
Objective: To establish the HPLC fingerprint of Bufonis Venenum pulp and the determination methods of eight components in Bufonis Venenum pulp, and compare the differences of components of Bufonis Venenum pulp in different origins for quality evaluation. Methods: The mobile phase was acetonitrile and water with a gradient elution program. The detection wavelength was set at 296 nm. The flow rate was 1.2 mL/min. The column temperature was set at 30 ℃, and 10 μL of the test solution was injected. HPLC fingerprint of Bufonis Venenum pulp was established and eight components were determined. The results were analyzed by cluster analysis and principal component analysis. Results: There were 12 common peaks in the HPLC fingerprints of 18 batches of samples, and the similarity of each sample was different. The linear relationship of eight components was good (r2 > 0.999 5), RSD of precision and repeatability was less than 0.5%, and the stability was also good within 30 h (RSD < 0.7%). The average recoveries of gamabufotalin, arenobufagin, telocinobufagin, bufotaline, cinobufotalin, bufalin, cinobufagin and resibufogenin were 103.7%, 103.0%, 102.9%, 103.0%, 103.9%, 100.3%, 103.4%, and 103.2% respectively, and RSD was all less than 1.2%. The results of the content determination, cluster analysis and principal component analysis of eight components showed that Bufonis Venenum pulp in different habitats were different from each other. Conclusion: The method is simple and reliable, which can provide some basis and reference for quality control of Bufonis Venenum pulp.
ABSTRACT
Objective: To optimize the extraction and purification technology of liposoluble constituents from Bufonis Venenum. Methods: The total content of cinobufagin and resibufogenin was taken as the index, and the ethanol potency, solvent multiplication and extraction time were taken as the investigation factors. The optimum extraction process parameters was determined by orthogonal test. Through single factor investigation experiment combined with Box-Behnken response surface method, taking the expansion agent ratio, ratio of diameter to height, adsorbent and sample quantity as the investigation factors, the optimum purification process was selected and determined. Results: It was determined that the optimum extraction process for the addition was 10 times 85% ethanol for 90 minutes’ extraction, the best purification process for the expansion agent cyclohexane-chloroform- acetone was 4:3:3, column height and diameter was 7:1, adsorbent and sample volume was 5.5:1. The content of two kinds of toad poison base purified was up to 66.51%. Through the verification of three batches of amplification process, it was shown that the model fit well, and the separation and purification of toad poison ligand by silicone column chromatography had the advantages of simple operation, fast separation speed, and good separation effect. Conclusion: The selected process is reasonable and feasible, which provides technical reference and basis for the industrialization application of the product in the future.
ABSTRACT
Objective: To study the constituents and metabolites in rat brain after ig administration of Xueshuan Xinmaining Tablet (XXT) based on the serum medicinal chemistry. Methods: The acute blood stasis rat model was replicated by ice bath-adrenalin method, and XXT [1.4 (g∙kg-1∙d-1)] was administered orally for 8 d. The brain samples were collected and pretreated after ig administration for the last time. Then the absorbed prototype constituents and their metabolites in rats plasma were rapidly analyzed and identified by combination of ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and multivariate statistical analysis. Results: Finally, a total of 11 absorbed prototype constituents and three metabolites were identified, including phenanthrene, bufonis venenum oxadiene, and other structural types of compounds. These constituents absorbed into brain may be the potential bioactive components in XXT. Conclusion: The establishment of UPLC-Q-TOF/MS analysis method explained comprehensively the brain migration component of XXT, and provided scientific basis for clarifying its substance basis of pharmacodynamics of this large variety of Chinese medicine.
ABSTRACT
As known,simultaneous determination of various chemical indicators is one of the future trends in quality control of traditional Chinese medicines because of the extremely complex chemical compositions. This project is to screen the quality markers that can accurately control the quality of the Bufonis Venenum by exploring the intrinsic correlation of components. In this study,venom of Bufo bufo gargarizans from 17 different sources were used as research samples,and the contents of 7 bufogenin were determined by HPLC-DAD. Then,the data obtained were analyzed by Spearman correlation analysis and principal component analysis( PCA). In addition,a stepwise regression analysis was used to establish a predictive model for the contents of the seven bufogenin components( independent variable) and the total contents of the bufogenin( dependent variable). The results indicated that there is a significant positive correlation between the contents of telocinobufagin and cinobufotalin,and there is a significant positive correlation between the contents of bufalin,cinobufagin and resibufogenin. In contrast,the contents of telocinobufagin and cinobufotalin are negatively correlated with the contents of bufalin,cinobufagin and resibufogenin. However,the correlation between gamabufotalin and bufotalin and other components are not obvious. Furthermore,further study found that there is a correlation between the sum of the contents of bufalin,cinobufagin and telocinobufagin and the total contents of the bufogenin. In fact,the application of bufalin,cinobufagin and telocinobufagin as the quality control indicators of the Bufonis Venenum can better reflect the quality characteristics of the Bufonis Venenum compared with the previous quality control indicators. The conclusions will provide a reference for the revision of the quality standards of the Bufonis Venenum.
Subject(s)
Animals , Amphibian Venoms , Chemistry , Bufanolides , Bufo bufo , Chromatography, High Pressure Liquid , Medicine, Chinese Traditional , Quality ControlABSTRACT
Objective To study the changes of chemical constituents and pharmacodynamics with different drying methods (sun- drying, 50 ℃ vacuum-drying, 50 ℃ and 80 ℃ heat-drying, freeze drying method) in Bufonis Venenum. Methods HPLC method and TLC method was established for studying the changes of chemical constituents from B. Venenum before and after being dried, and determine the inhibitory effect of dried samples on five different tumor cell lines proliferation by MTT assay. Results The B. Venenum processed by five different methods were different in character, while no significant differences in the types and content of chemical constituents; The total content of resibufogenin and cinobufagin was more than 6%, which was consistent with the 2015 edition of Chinese Pharmacopeia; 50 ℃ and 80 ℃ heat-drying of B. Venenum showed more effective inhibitiory effect than other dry methods. Conclusion The appearance of B. Venenum met the criterion of pharmacoperia by sun-drying and 50 ℃ heat-drying method. Although the color of B. Venenum under the vacuum-drying and freeze drying method did not meet the requirements of Chinese Pharmacopeia, the main effective components have a few changes.
ABSTRACT
Bufonis Venenum, as a precious Chinese materia medica, has complex chemical constituents and has been widely used in clinical treatment with significant effects. The chemical constituents in Bufonis Venenum mainly included bufadienolides, indole alkaloids, and steroids, etc. Modern pharmacological research has demonstrated its antitumor, cardiac, anti-inflammatory, and narcotic analgesic, etc. This paper reviewed the chemical constituents, pharmacological activity, and artificial synthesis of Bufonis Venenum, providing theoretical reference for material basic research, pharmacodynamic mechanism interpretation, development, and utilization.
ABSTRACT
Objective: To establish an HPLC-MS method for determining nine bufadienolides (gamabufotalin, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, cinobufagin, and resibufogenin) in Bufonis Venenum and Liushen Pill. Methods: HPLC analysis was performed on ODS-2 column (250 mm×4.6 mm, 5 μm) with the mobile phase made up of acetonitrie and 0.15% phosphoric in water. Flow rate was set at 1 mL/min and column temperature was 40℃. The detection wavelength was 296 nm. Nine bufadienolides were determined by HPLC-ESI-TOF-MS with the mobile phase made up of acetonitrie and 0.2% formic acid in water. Eletrospray ionization source was applied and operated in the positive ion mode. Results: Nine bufadienolides were separated at baseline with good linearity (r≥0.9996). The recovery rates were 92%-105% (RSD<5%). Conclusion: The method established is rapaid, simple, and accurate, which is helpful to control the quality of Liushen Pill.
ABSTRACT
Bufonis Venenum, as an important Chinese traditional natural medicine, has complex chemical composition and has been widely used in clinical treatment with significant effects. The chemical constituents in Bufonis Venenum mainly included bufadienolides, indole alkaloids, steriods, etc. With the further research on Bufonis Venenum both at home and abroad, its chemical composition and mechanism of pharmacological activities have been more and more clearly, especially in the aspects of narcotic analgesic, cardiac, and antitumor therapy. This paper reviewed the chemical composition and pharmacology advances of Bufonis Venenum in order to provide reference to clinical use.
ABSTRACT
Objective To assess the consistency of 30 batches of Xueshuan Xinmaining Tablets (XXT), we establish the UPLC-PDA fingerprint of XXT and provide reliable scientific basis for the Chinese medicine quality consistency. Methods Methanol was used to extract the XXT samples, and the extracts were measured by UPLC with the absorption wavelength at 280 nm. Waters Acquity UPLC HSS T3 reversed-phase column (50 mm × 2.1 mm, 1.8 μm) was used for determining the extracts at a flow rate of 0.5 mL/min and column temperature of 40 ℃. The mobile phase condition was acetonitrile-0.1% formic acid aqueous solution with gradient mode. The methodology, similarity calculation and cluster analysis of 30 batches of XXT samples were investigated. The common peak of XXT was belonged to single medicinal materials and common peaks were identified by comparing with the reference. Results The UPLC fingerprint of XXT was established. Twenty-seven common fingerprint peaks were indicated with wavelength 280 nm, Among them, peaks 1, 10, 12-16, 18, and 21-28 belonged to Salviae Miltiorrhizae Radix et Rhizoma (SMRR), peaks 2-4, 6, 8, 9, and 17 belonged to Chuanxiong Rhizoma (CR), peaks 3, 7, 9, 11, and 16 belonged to Ilicis Pubescentis Radix (IPR), peak 5 belonged to Sophora Flos (SF), peaks 19 and 20 belonged to Bufonis Venenum (BV). Peaks 4, 5, 13, 19, 20, and 28 were ferulic acid, rutin, salvianolic acid B,resibufogenin, cinobufagin, tanshinone IIA, respectively, compared with reference. The analyzed samples were classified into two classes and the similarity was greater than 0.960. Conclusion The method of XXT UPLC-PDA fingerprint was established with good precision, stability and repeatability, supplying helpful information for the comprehensive evaluation of XXT.
ABSTRACT
Bufonis Venenum is made by toad skin gland secretion after drying. As a kind of Chinese materia medica (CMM), it contains complex chemical composition and has these pharmacological effects: to remove toxic heat, wake up the brain orifices, eliminate the mass, step-down blood pressure, increase myocardial contraction force and coronary artery blood flow, and use for antitumor and anesthesia. It is widely used in diseases in clinic such as cardiovascular, respiratory, facial features, and surgical treatment. While, as toxic CMM, the concentration-response relationship, toxic ingredients, toxic dose, and poisoning mechanism should not be ignored also. The author preliminarily summaries its effects from composition, pharmacological effects and mechanisms, adverse reactions, processing technology, and pharmacokinetics, and provides the references for its further pharmacological study and compound preparation development.
ABSTRACT
Objective: To obtain antitumor active compounds with lower toxicity from main ingredients of Chinese materia medica Bufonis Venenum. Methods: Taking inhibition of in vitro tumor cells as active indexes, a bioassay-guided separation and toxicity evaluation in mice of ingredients in Bufonis Venenum were applied. Results: Bufotalin was seperated from Bufonis Venenum with significant inhibition and lower acute toxicity by active tracking method. The inhibitory effect on the proliferation of A549 was better, LD50 of bufotalin by iv administration to mice is about four times of bufalin, and bufotalin is stable. Conclusion Integrated the activity, toxicity, and stability factors of the compounds, bufotalin from Bufonis Venenum will deserve further research than any other similar compounds in potential antitumor activity.